Studies On ?-Lactamase Expression Induced By Cell Wall-Targeting Antibiotics In Shewanella Oneidensis

?-Lactamase can hydrolyze?-lactam antibiotics??-lactams?.The induction of?-lactamase expression is the most important reason for?-lactam resistance in Gram-negative bacteria.By binding to penicillin binding proteins?PBPs?covalently,?-lactams prevent bacterial cell wall synthesis,eventually resulting in the induction of?-lactamase expression.In addition to?-lactams,there are a variety of antibiotics that specifically target the bacterial cell wall biosynthesis,such as vancomycin,D-cycloserine and moenomycin.However,it remains unclear whether these antibiotics can also induce the expression of?-lactamase.Shewanella oneidensis is a Gram-negative bacterium that widely distributed in environment.This bacterium is naturally resistance to?-lactams.Previous studies have shown that the?-lactamase gene bla A is induced by ampicillin?a kind of?-lactam?in S.oneidensis.This study investigated the effects of various cell wall-targeting antibiotics on the expression of bla A gene and its molecular mechanism.The results are as follows:By measuring the effect of?-lactams on the activity of the bla A promoter(P_{bla A}),it was found that different?-lactams had different ability to induce the expression of bla A.Cefsulodin and cephalothincan drastically enhanced bla A expression,while cefotaxime and aztreonam failed to induce bla A expression.Interestingly,other types of cell wall-targeting antibiotics,such as vancomycin,D-cycloserine and moenomycin,also induced the expression of bla A.Especially,vancomycin and D-cycloserine significantly enhanced the resistance to ampicillin.In contrast,other antibiotics and chemicals targeting cell membrane had no effect on bla A expression.These results indicated that the bla A gene was not only induced by?-lactams but also induced by other cell wall-targeting antibiotics.The induction of?-lactamase expression is usually involved in the inactivation of the targets?PBPs?of?-lactams.S.oneidensis contains at least eight PBPs.Previous studies had found that PBP1a may be the primary target for?-lactams.By sequence comparison,this study found that S.oneidensis PBP1a(^SoPBP1a)has 50%homology with Escherichia coli PBP1a(^EcPBP1a).^SoPBP1a also contained identical domains and signature motifs to ^EcPBP1a.^SoPBP1a activity could be functionally substituted by ^EcPBP1a.^SoPBP1a is a bifunctional enzyme harboring both transglycosylase and transpeptidase activities.^SoPBP1a variants lacking either activity enhanced the expression of bla A and the resistance to ampicillin,and also result in bacterial cell wall damage.Subsequent research found that cell wall-targeting antibiotics had no effect on ^SoPBP1a expression,indicating that bla A expression was not derived from the inactivation of^SoPBP1a.However,cell wall-targeting antibiotics that induce bla A expression could cause cell wall damage.Further studies found that a two-component system Sbr KR was activated by these cell wall-targeting antibiotics.Sbr K is a histidine kinase located in the cytoplasmic membrane,while Sbr R is a response regulator located in the cytoplasm.Cell wall-targeting antibiotics failed to induce bla A expression and to enhance ampicillin resistance when loss of either Sbr K or Sbr R.Similar results are obtained when the conserved phosphorylation sites of Sbr KR were substituted with alanine.It can be concluded that cell wall-targeting antibiotics induced the expression of bla A gene through the two-component system Sbr KR,which was dependend on the phosphorylation of Sbr KR.Bioinformatics analysis showed that the periplasmic domain of Sbr K was a carbohydrate-binding module.The expression of bla A was no longer induced by single amino acid substitutions?R177A,Y178A and I289A?in the putative periplasmic binding pocket of Sbr K,indicating that these sites played an important role in binding with unknown ligands.In conclusion,cell wall-targeting antibiotics with different structures may produce a common ligand,which binds to Sbr K and then activates Sbr KR,eventually inducing the expression of?-lactamase.